Extraction and assay conditions for β-glucosidase from propolis were optimized. Highest enzyme activity was obtained in a citric acid-disodium hydrogen phosphate buffer at pH 6.0 with 2.5% insoluble polyvinylpyrrolidone at incubation temperature of 57 °C. β-Glucosidase activities were found in all freshly harvested propolis while β-glucosidase activities were scarcely present in the randomly bought propolis. Propolis was stored at –20 °C and 4 °C for 3 mo with almost no loss of β-glucosidase activity, but at room temperature the activity decreased exponentially with the increase of storage time.

These results indicated that the activity of β-glucosidase could be a candidate for propolis-freshness index. β-Glucosidase from propolis was capable of hydrolyzing p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-galactoside, but lacked activity toward p-nitrophenyl-β-D-glucuronide, p-nitrophenyl-β-D-cellobioside, amygdalin, cellobiose, and gentiobiose. These results were consistent with the hypothesis that flavonoid glucosides were hydrolyzed by β-glucosidase during propolis collection and processing and provided a possible explanation for why some flavonoid biosides (that is, rutin and isorhamnetin-3-O-rutinoside) exist in propolis.

Practical Application: β-Glucosidase activity was detected and partial characterization of the enzyme was determined in propolis. The enzyme activity decreased exponentially with the increase of storage time at room temperature, which suggested that the activity of β-glucosidase could be regarded as a freshness index of propolis. The research will be useful for studying the chemical constituents of propolis.